Integrins are cell surface receptors that mediate cell to cell, or cell to extracellular matrix adhesion. They play a major role in the regulation of immune cell recruitment to inflamed endothelia and sites of inflammation. Integrins participate in antigen presenting cell-lymphocyte interactions, retention and mobilization of immature progenitors in the bone marrow, cancer cell trafficking, metastasis and other events. They represent a target for several existing drugs for treatment of inflammatory diseases, anti-angiogenic therapy, and anti-thrombotic therapy, among others. Integrin ligands can also be used as imaging tools. Integrin dependent adhesion avidity is regulated by a number of conformational changes of the protein. These can occur without a significant change in the expression levels of the molecules. Conformational changes include an increase in the affinity of the ligand binding pocket, and others, that consists of the unbending (extension) of the integrin, along with hybrid domain swing, as well as integrin “leg” separation. Recent data suggest that at least some of these conformations are regulated independently from the others. Conformational changes can be detected using conformationally sensitive antibodies, which bind to defined epitopes exposed only in certain molecular conformations. Some of these are known to be induced by the binding of the ligand (so called ligand induced binding sites (LIBS)). Several antibody epitopes have been mapped to the part of the VLA-4 integrin surface between α and β1-subunits, which is hidden in the resting, low affinity state because of the close subunit proximity, and exposed upon activation and/or ligand binding. The integrin conformation with exposed epitopes is attributed to the high affinity activation state in one model of integrin activation and the ligand occupied conformation according to another. However, despite differing opinions about the role of epitope exposure, epitope exposure represents a valuable tool for monitoring integrin conformations using flow cytometery techniques.
Discovery of new small molecules that bind to the integrin ligand binding site and block interaction with its natural ligand, is part of the ongoing drug discovery process. The ability to detect specific binding of the ligand and determine its binding affinity is critical for these approaches. In this case a desirable assay would be if the binding of the unlabeled small molecule could be detected in a homogeneous assay. Here we describe a novel approach for the detection of the ligand binding affinity based upon induction of ligand induced epitopes. Using commercially available conformationally sensitive mAbs we were able to confirm induction of ligand induced epitopes, as well as ligand binding affinity for two previously described VLA-4 integrin ligands. EC50 values for the conformational mAb binding showed a good correlation with Kis determined in the competitive binding assay with a well characterized fluorescent ligand. We have also determined binding constants for two novel VLA-4 ligands, and verified them using a competitive binding assay. Ligands which induce activation epitopes may formally be referred to as agonists if they also induce intracellular signaling. This current approach can be extended to other integrins, and can be adapted for a high-throughput flow cytometry format.
From a therapeutic point of view integrins are the most important class of cell adhesion receptors that mediate cell to cell, or cell to extracellular matrix adhesion. VLA-4 (very late antigen 4, α4β1-integrin, CD49d/CD29) plays a major role in the regulation of immune cell recruitment to inflamed endothelia and sites of inflammation. It participates in antigen presenting cell-lymphocyte interactions, retention and mobilization of immature progenitors in the bone marrow, cancer cell trafficking, metastasis and other events. Integrins represent an attractive target for several existing drugs for treatment of inflammatory diseases, anti-angiogenic therapy, and anti-thrombotic therapy, among others as described herein. Integrin ligands can also be used as imaging tools, as well as probes for studies of integrin functional activity and molecular conformation.
Multiple small molecules have been developed in an attempt to regulate integrin dependent adhesion. Competitive antagonists can bind to the natural ligand binding pocket, and block interaction between integrins and integrin natural ligands. Because the binding pocket is located between the α subunit and β subunit I-like domain they also are termed α/β I-like competitive antagonists. Multiple compounds of this type were developed for αIIbβ3, αvβ3, and α4β1 integrins. Several integrins have an additional domain that is inserted within α-subunit β-propeller (A domain or I domain), which is evolutionarily related to the β I-like domain. The I domain serves as a ligand binding site for these integrins. Two types of allosteric antagonists for these integrins have been described: α/β I-like allosteric antagonists and α I allosteric antagonists. Previously, no allosteric antagonists have been identified for non I domain containing integrins (such as VLA-4). Using the method described in the invention we were first to identify VLA-4 integrin allosteric antagonists (Chigaev, et al., J Biol Chem. 286, 5455-63 (2011)).
One of the features of integrin competitive antagonists is to occupy the ligand binding pocket and induce a conformational change that is similar to the conformational change induced by a natural ligand. Recently, the present inventors showed that this feature can be used for the identification of unknown integrin antagonists, and determination of the ligand binding affinity for unlabelled small integrin ligands, See, Njus, et al., Assay. Drug Dev. Technol. 7, 507-515 (2009) and Chigaev, et al., J. Biol. Chem. 284, 14337-14346 (2009), Chigaev, et al., J Biol Chem. 286, 5455-63 (2011).
We have modified the existing assay to specifically detect only VLA-4 allosteric antagonists, and performed high-throughput flow cytometry-based screen of the Prestwick Chemical Library (PCL), which represents one of “smart screening libraries” designed to decrease a number of “low quality” hits.
In the present application, several structurally related compounds that were able to prevent exposure of ligand induced binding sites (LIBS) after the addition of VLA-4 specific ligand, decrease binding affinity of VLA-4 specific ligand, and block VLA-4/VCAM-1 dependent cell adhesion are reported. Because these compounds are previously used or currently marketed drugs, which are known to possess immunosuppressive properties that are specifically attributed to the cell mediated component (4), this effect upon VLA-4 ligand binding provides a plausible explanation for the mechanism of immunosuppression.